Hepatitis C virus (HCV) is the major etiological agent of post-transfusion and community-acquired non-A non-B hepatitis worldwide. It is estimated that over 200 million people worldwide are infected by the virus. A high percentage of carriers become chronically infected and many progress to chronic liver disease, or so-called chronic hepatitis C. This group is in turn at high risk for serious liver disease such as liver cirrhosis, hepatocellular carcinoma and terminal liver disease leading to death.
The mechanism by which HCV establishes viral persistence and causes a high rate of chronic liver disease has not been thoroughly elucidated: It is not known how HCV interacts with and evades the host immune system. In addition, the roles of cellular and humoral immune responses which protect against HCV infection and disease have yet to be established.
HCV is an enveloped positive strand RNA virus of the Flaviviridae family. The single strand HCV RNA genome is of positive polarity and comprises one open reading frame (ORF) of approximately 9600 nucleotides in length, which encodes a linear polyprotein of approximately 3010 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce structural and non-structural (NS) proteins. The structural proteins (C, E1, E2 and E2-p7) comprise polypeptides that constitute the virus particle. The non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B) encode for enzymes or accessory factors that catalyze and regulate the replication of the HCV RNA genome. Processing of the structural proteins is catalyzed by host cell proteases. The generation of the mature non-structural proteins is catalyzed by two virally encoded proteases. The first is the NS2/3 zinc-dependent protease which auto-catalyzes the release of the NS3 protein from the polyprotein. The released NS3 protein contains an N-terminal serine protease domain and catalyzes the remaining cleavages from the polyprotein. The released NS4A protein has at least two roles. The first role is to form a stable complex with NS3 protein and assist in the membrane localization of the NS3/NS4A complex. The second role of the NS4A protein is to act as a cofactor for NS3 protease activity. This membrane-associated complex in turn catalyzes the cleavage of the remaining sites on the polyprotein, thus effecting the release of NS4B, NS5A and NS5B. The C-terminal segment of the NS3 protein also harbors nucleoside triphosphatase and RNA helicase activity. The function of the NS4B protein is unknown. NS5A is a highly phosphorylated protein that appears to be responsible for the interferon resistance of various HCV genotypes. NS5B is an RNA-dependent RNA polymerase (RdRp) that is involved in the replication of HCV.
The open reading frame of the HCV RNA genome is flanked on its 5′ end by a non-translated region (NTR) of approximately 340 nucleotides that functions as the internal ribosome entry site (IRES), and on its 3′ end by an NTR of approximately 230 nucleotides. Both the 5′ and 3′ NTRs are important for RNA genome replication. The genomic sequence variance is not evenly distributed over the genome and the 5′NTR and parts of the 3′NTR are the most highly conserved portions.
The cloned and characterized partial and complete sequences of the HCV genome have been analyzed with regard to appropriate targets for prospective antiviral therapies. The following four viral enzyme activities provide possible targets: (1) the NS2/3 protease; (2) the NS3/4A protease complex, (3) the NS3 helicase and (4) the NS5B RNA-dependent RNA polymerase (NS5B RdRp). The NS3 protease has also been crystallized to reveal a structure reminiscent of other serine proteases (Love et al., 1996; Kim et al. 1996).
NS3 protease activity is an attractive target for drug discovery. Enzymatic studies have shown that peptides based on the N-terminal product of the NS5A/5B cleavage site are competitive inhibitors of the enzyme. These peptides have served as a useful starting point in medicinal chemistry efforts to rationally design NS3 protease inhibitors as clinically effective anti-HCV compounds.
Chronic hepatitis C has emerged as an important clinical indication, an effective treatment for which has yet to be developed due to poor response rates to currently existing treatments. For example, the newly approved standard treatment, pegylated-interferon in combination with Ribavirin, exhibits a sustained response rate of 40 to 50%. However, a majority of patients still do not elicit a sustained anti-viral response, particularly against the interferon-resistant HCV genotypes, 1a and 1b.
WO 00/09543, WO 00/09558 and WO 00/59929 (all three incorporated herein by reference) disclose certain types of inhibitors of the HCV NS3 protease that are highly active and selective. These compounds have potential for becoming the next generation of anti-HCV treatment. It can be expected that these inhibitors, as well as many other antiviral treatments, will eventually give rise to viruses that are at least partially resistant to such inhibitors.
Knowledge of mutations which render HCV resistant to inhibitors provides the basis for identifying inhibitors that are effective against such resistant strains. Trozzi et al., 2003 has disclosed a resistant mutant replicon having three individual amino acid substitutions (D168A/Y/V) that render the protease resistant to an inhibitor of the NS3 protease.
Accordingly, in an effort to develop a treatment with long-term efficacy that suppresses or overcomes anti-HCV resistance, we describe a means to identify anti-HCV compounds that exhibit activity against inhibitor resistant HCV strains.